A new protecting group for tryptophan in solid-phase peptide synthesis which protects against acid-catalyzed side reactions and facilitates purification by HPLC

Article abstract of DOI:10.1016/j.tetlet.2009.04.014

The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting derivative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into

Full text of DOI:10.1016/j.tetlet.2009.04.014

Tetrahedron Letters 50 (2009) 2976–2978
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Tetrahedron Letters
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A new protecting group for tryptophan in solid-phase peptide synthesis
which protects against acid-catalyzed side reactions and facilitates
purification by HPLC
*
Karolina Wahlström, Anders Undén
Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The indole nucleus of Z-Trp-OBzl is modified by acylation of the indole nitrogen using Boc-N-methyl
butyric acid followed by catalytic hydrogenation and introduction of the Fmoc group. The resulting deriv-
ative, Fmoc-Trp(Boc-Nmbu)-OH, is incorporated into peptide chains via solid-phase peptide synthesis
(SPPS). After assembly of the peptide chain, the Boc group is cleaved by treatment with TFA. The peptide
is isolated with the tryptophan residue modified with a cationic 4-(N-methylamino) butanoyl group,
which improves the solubility of the peptide during HPLC purification. On treatment of the purified pep-
tide at pH 9.5, the Nmbu group undergoes an intramolecular cyclization reaction; this results in the fully
deprotected peptide and N-methylpyrrolidone.
Received 15 January 2009
Revised 23 March 2009
Accepted 3 April 2009
Available online 9 April 2009
Keywords:
Solid-phase peptide synthesis
Protecting group
HPLC
Ó 2009 Elsevier Ltd. All rights reserved.
Tryptophan
Fmoc amino acids
Highly hydrophobic peptides, especially those prone to b-sheet
formation, can cause significant problems in the handling of a pep-
tide after cleavage from the resin.1 These peptides tend to be diffi-
cult to dissolve in the solvents typically used for HPLC purification,
hence, only small amounts can be loaded onto the column and the
peptides often elute as broad peaks with poor resolution.2
Several studies have shown that these problems can be circum-
vented by adding charged residues to the peptides, especially argi-
nine residues as a ‘solubilizing tail’.3–6 These residues are present
during the purification but are cleaved later in a separate step.
The efficiency of this approach has been demonstrated in several
reports, but this method is dependent on the efficient and mild
cleavage of additional cationic residues after purification of the
peptide.
In two recent publications, we have suggested an alternative
strategy to increase the solubility of synthetic peptides during
the purification step.7,8 In these studies, the phenolic alcohol
groups on tyrosine residues or the peptide backbone-protective
N-(2-hydroxy-4-methoxybenzyl) (Hmb) group were modified with
the Boc-N-methyl-N-[(2-methylamino)ethyl]carbamoyl group
(Boc-Nmec) during the synthesis. After cleavage of the peptide
from the resin with TFA, the Boc group was removed while the cat-
ionic N-methyl-N-(2-methylamino)ethyl carbamoyl group was still
attached to the peptide, thereby increasing the solubility and facil-
itating purification. By exposing the peptide to slightly alkaline pH,
the Nmec group is cleaved via an intramolecular cyclization, giving
the fully deprotected peptide. In this study, we propose an analo-
gous strategy for tryptophan.
Tryptophan is a very hydrophobic residue and it can therefore
contribute to reducing the solubility of a peptide during purifica-
tion steps. The indole ring of tryptophan is also susceptible to
alkylation by cations formed during acidolytic cleavage of protect-
ing groups, these side reactions can be suppressed by addition of
scavengers or by the use of electron-withdrawing protecting
groups on the indole nucleus. In the present study, we report the
use of a new protecting group, the 4-(N-methylamino)butanoyl
group (Nmbu) for tryptophan (1) that provides protection against
modification by reactive cations during the cleavage reaction and
at the same time facilitates purification by supplying additional
cationic charges during the purification step (Scheme 1).
Esters of 4-aminobutyric acid are known to undergo intramo-
lecular lactamization, giving pyrrolidone and an alcohol.9 We
therefore wanted to investigate whether indolyl derivatives of N-
methyl-4-amino butyric acid could be cleaved by intramolecular
aminolysis at a rate that is sufficiently fast to be of practical use
in solid-phase peptide synthesis.
The synthesis of Fmoc-Trp(Boc-Nmbu)-OH is outlined in
Scheme 2. The indole nitrogen of Z-Trp-OBzl (4) was acylated as
described by Klausner et al. and Snider and Zeng using the p-nitro-
phenyl ester of Boc-N-methyl-4-amino butyric acid (5) and KF/
18-crown-6, in 67–87% yield.10,11 The fluoride anion mediated
* Corresponding author. Tel.: +46 816 41 17; fax: +46 816 13 71.
E-mail address: andersu@neurochem.su.se (A. Undén).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.014

K. Wahlström, A. Undén / Tetrahedron Letters 50 (2009) 2976–2978
2977
O
H
N
O
O
H
N
H
N
R1
R2
N
R2
N
R1
R2
NH
R1
H
N
H
NH
O
O
1
2
3
Scheme 1. General scheme for the use of 4-(N-methylamino)butanoyl (Nmbu)-
protected tryptophan in SPPS. After cleavage from the resin, the peptide is isolated
with the cationic Nmbu group attached to the tryptophan residue of 1; R1,
R
2 = peptide chains. In this form, the peptide has improved solubility and can be
purified by reverse-phase HPLC. After purification, the peptide is exposed to
alkaline pH during which the Nmbu group is cleaved via an intramolecular
cyclization reaction as shown in 2 to give the fully deprotected peptide 3.
Z
HN
Z
HN
O
O
OBzl
NH
OBzl
N
O2N
a
Boc
N
O
Figure 1. Stability of the Boc-Nmbu group to piperidine treatment. The peptide
Fmoc-Lys(Boc)-Trp(Boc-Nmbu)-Leu-Pro-Phe-Leu-Ala attached to a solid phase was
exposed to 20% piperidine in DMF for 18, 24 or 48 h. The peptide was then cleaved
from the resin with a mixture of TFA (95%), triisopropylsilane (2.5%) and water
(2.5%) and was analyzed by reverse-phase HPLC at 215 nm.
Boc
N
O
O
4
6
5
Fmoc
HN
O
O
H2N
b
c
OH
OH
N
N
Boc
Boc
N
N
alkylation of tryptophan and can be removed by nucleophiles such
as piperidine or, under acidic conditions, by ethanedithiol.13
Although the formyl protecting group is very sensitive to nucleo-
philes, studies have shown that other acyl derivatives are much
more stable. Arai and co-workers used indole as a protecting group
for carboxylic acids and cleavage required 3 M NaOH in methanol
for 2.5–35 h for complete deprotection, depending on the structure
of the carboxylic acid.14
O
O
7
8
Scheme 2. Synthesis of Fmoc-Trp(Boc-Nmbu)-OH. Reagents (a) KF/18-crown-6/
N,N0-diisopropylethylamine, THF; (b) H2/10% Pd on charcoal, EtOH; (c) (i) Trimeth-
ylsilyl chloride, (ii) N,N0-diisopropylethylamine, Fmoc-Cl, MeCN.
reaction was carried out in dry tetrahydrofuran instead of dry ace-
tonitrile, as was recommended by Klausner and co-workers. In our
hands, no product was obtained with acetonitrile as solvent. The
purified product 6 was hydrogenated with H2/Pd, to remove the
To investigate the stability of Boc-Nmbu to piperidine treat-
ment, Fmoc-Trp(Boc-Nmbu)-OH 8 was incorporated into the mod-
el peptide Fmoc-Lys(Boc)-Trp(Boc-Nmbu)-Leu-Pro-Phe-Leu-Ala
attached to a Wang resin, and was treated with 20% piperidine in
DMF for 18, 24 or 48 h, followed by treatment with TFA. As can
be seen in Figure 1, only small amounts of the Nmbu group were
cleaved after 48 h, showing that the Boc-Nmbu group is sufficiently
stable to be used even for large peptides in Fmoc SPPS.
Cleavage of the Nmbu group under aqueous alkaline conditions
was studied by exposing the peptide H-Lys-Trp(Nmbu)-Leu-Pro-
Phe-Leu-Ala-OH to Tris buffer (Fig. 2). It was found that the rate
of cleavage was slow below pH 9.0 but proceeded with a half-life
of about 5–7 min at pH 9.5, as measured by absorption at
240 nm. The reaction mechanism was (at least partly) an intramo-
benzyloxycarbonyl and the benzyl protecting groups. The a-nitro-
gen of H-Trp(Boc-Nmbu)-OH (7) was protected with Fmoc-Cl as
described by Bolin and co-workers, and the product was purified
by flash chromatography giving Fmoc-Trp(Boc-Nmbu)-OH (8) in
97% yield.12 The 1H NMR and 13C NMR spectra were in accordance
with the expected structure. A detailed description of the synthesis
and the NMR data are given in Supplementary data.
The Nmbu group is an acyl type protecting group, and in this re-
spect it is similar to the nucleophile-sensitive formyl group used to
protect tryptophan in Boc chemistry. The formyl group prevents
Figure 2. HPLC profiles, monitoring the cleavage of Nmbu-protected tryptophan in the peptide H-Lys-Trp(Nmbu)-Leu-Pro-Phe-Leu-Ala-OH exposed to Tris buffer, pH 9
(a) H-Lys-Trp(Nmbu)-Leu-Pro-Phe-Leu-Ala-OH; (b) H-Lys-Trp-Leu-Pro-Phe-Leu-Ala-OH after 90 min showing complete deprotection of tryptophan; (c) N-methylpyrrolidone.

2978
K. Wahlström, A. Undén / Tetrahedron Letters 50 (2009) 2976–2978
10000
7500
5000
2500
0
We conclude that the Nmbu protecting group reported in this
pH 9.5
pH 9
pH 8.5
study, together with the previously reported Nmec protecting
group for tyrosine and the Hmb group, can be useful tools in the
synthesis of very hydrophobic peptides such as transmembrane
segments of membrane proteins. By using these protecting groups,
peptides with tryptophan, tyrosine and glycine residues can be
purified as cationic peptides after cleavage, even if the sequence
contains no cationic residues.
Acknowledgement
0
1000
2000
3000
4000
t(sec)
We are grateful to Anders Florén at the department of Neuro-
chemistry, Stockholm University for his kind advice and guidance
in the kinetics studies of the Nmbu group cyclization by fluores-
cence spectroscopy.
Figure 3. Kinetics of the cleavage of the Nmbu group in the peptide H-Lys-
Trp(Nmbu)-Leu-Pro-Phe-Leu-Ala-OH as measured by the increase in fluorescence
(F/F0) at 360 nm and at different pH values.
Supplementary data
lecular cyclization reaction, as HPLC analysis at 215 nm showed
that a product was formed which had the same elution time as
N-methylpyrrolidone.
Supplementary data (experimental details for the synthesis of
Fmoc-Trp(Boc-Nmbu)-OH and 13C NMR and 1H NMR for Fmoc-
Trp(Boc-Nmbu)-OH) associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.04.014.
A more accurate determination of the kinetics of intramolecular
cyclization can be obtained by fluorescence spectroscopy (Fig. 3).
The indole ring of tryptophan emits fluorescence at 353 nm whilst
the Nmbu-protected indole ring does not.15 Thus, the formation of
deprotected tryptophan can be measured as an increase in the
intensity of fluorescence. As expected, the rate of cyclization was
pH-dependent and the half-life of Nmbu-protected tryptophan
was 4–5 min at pH 9.5. However, care must be taken to extrapolate
these values to reaction conditions in which the peptide is prone to
aggregation and is present in much higher concentration during
the cyclization reaction. Under these conditions, the reaction rate
can be expected to be significantly slower.
During treatment with TFA, tryptophan can undergo oxidation,
dimerization and alkylation through reactive cations formed dur-
ing cleavage of the peptide from the resin.13 In Fmoc chemistry
using Nin-Boc protected tryptophan, these side reactions can be
suppressed to low levels.16–18 In order to compare the protection
provided with the Nmbu group, we synthesized peptides where
tryptophan was left unprotected, or protected by the Boc or the
Boc-Nmbu group. This was followed by cleavage with TFA/triiso-
propylsilane/H2O in a 95:2.5:2.5 ratio and the products were ana-
lyzed by HPLC and MALDI-TOF mass spectroscopy (data not
shown). Unprotected tryptophan resulted in the formation of high
levels of by-products while both Boc and Boc-Nmbu protection
suppressed these side reactions with similar efficiency.
References and notes
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